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WO2024254772A1 - Trophoblaste modifié et son utilisation - Google Patents

Trophoblaste modifié et son utilisation Download PDF

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Publication number
WO2024254772A1
WO2024254772A1 PCT/CN2023/100058 CN2023100058W WO2024254772A1 WO 2024254772 A1 WO2024254772 A1 WO 2024254772A1 CN 2023100058 W CN2023100058 W CN 2023100058W WO 2024254772 A1 WO2024254772 A1 WO 2024254772A1
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Prior art keywords
cells
cell
engineered
trophoblast
domain
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English (en)
Chinese (zh)
Inventor
苏俊
李陈梅
王爽
罗涛
章倩倩
任江涛
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Hongkong Bioheng Biotech Ltd
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Hongkong Bioheng Biotech Ltd
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Priority to PCT/CN2023/100058 priority Critical patent/WO2024254772A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of cell therapy. More specifically, the present invention relates to engineered trophoblastic cells and their uses.
  • NK cells Natural killer cells are important lymphocytes in the body, with cytotoxic and immunomodulatory functions, participating in innate immunity and adaptive immunity in the body, and are considered to be one of the most potential tumor-killing effector cells. NK cells express a variety of activation receptors, among which NKp46 is considered to be a unified characteristic molecule of NK cells across species.
  • NK cells have the following advantages in the field of cell therapy: 1. NK cells can spontaneously kill tumor cells without prior sensitization or antigen presentation, and take effect more quickly; 2. NK cells can kill tumor cells through multiple pathways; 3. NK cell therapy does not produce serious adverse reactions, only mild graft-versus-host disease (GvHD); 4. NK cells can be directly used for allogeneic universal immune cell therapy without modification, etc.
  • GvHD graft-versus-host disease
  • NK cells can be directly used for allogeneic universal immune cell therapy without modification, etc.
  • NK cells only account for 1%-32.6% of normal human peripheral blood lymphocytes, it is difficult to expand in vitro, and their survival lifespan in vivo is limited. NK cell immunotherapy has always been limited by the problem of not being able to obtain a sufficient number of cells.
  • cytokines such as IL-2 and IL-15 can be used to expand NK cells, but the effect is limited. Therefore, it is still necessary to develop an engineered trophoblast cell to study the function of NK cells and lay the foundation for cell therapy.
  • the purpose of the present invention is to provide an engineered trophoblast cell, which expresses a cell surface molecule that specifically recognizes NKp46. Compared with unengineered trophoblast cells, the engineered trophoblast cell is used to activate NK cells, which can enhance the NK cell proliferation ability and tumor cell killing activity.
  • the present invention provides an engineered trophoblast cell expressing a cell surface molecule that specifically recognizes NKp46, wherein the cell surface molecule comprises at least an antibody targeting NKp46 and a transmembrane domain.
  • the term "feeder cells” refers to cells that are added to target cell (e.g., NK cell) cultures to support Cells that maintain their survival and/or growth.
  • Trophoblasts provide complete and functional extracellular matrix and matrix-related factors, and secrete known and unknown cytokines into conditioned medium. Trophoblasts are usually in a state of growth arrest to prevent them from proliferating in culture, but maintain their survival. Growth arrest can be achieved by irradiation of an effective dose or treatment with an effective dose of a chemical substance (such as mitomycin C).
  • Trophoblasts suitable for the present invention include: PBMC, RPMI8866, HFWT, K562, EBV-LCL, 721.221 and NK92 cells, etc.
  • cell surface molecule refers to a molecule expressed on the cell surface that is capable of specifically binding to NKp46.
  • Such surface molecules generally include antibodies that are capable of specifically binding to NKp46 and transmembrane domains that anchor the surface molecule to the cell surface.
  • the surface molecule also includes a hinge region between the antibody and the transmembrane domain, an intracellular domain responsible for signal transduction, an exogenous cell activation molecule, and the like.
  • antibody refers to any structure or functional variant thereof that can bind to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, and can be selected from human antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, and functional fragments thereof.
  • antibodies include but are not limited to complete antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, sdAb, nanobodies, diabodies, anticalins, DARPINs, etc.
  • antibodies can be monovalent or divalent, and can be monospecific, bispecific, or multispecific antibodies.
  • Antibodies targeting NKp46 known in the art can be used in the present invention.
  • the antibody targeting NKp46 is selected from a complete antibody, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, diabody, sdAb and nanobody, preferably selected from Fab, scFv, single domain antibody and nanobody.
  • the antibody targeting NKp46 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO:7; wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO:8.
  • the CDR1-L is shown as SEQ ID NO: 1
  • CDR2-L is shown as SEQ ID NO: 2
  • CDR3-L is shown as SEQ ID NO: 3
  • CDR1-H is shown as SEQ ID NO: 4
  • CDR2-H is shown as SEQ ID NO: 5
  • CDR3-H is shown as SEQ ID NO: 6.
  • the light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO:7; the heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO:8.
  • the antibody targeting NKp4 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:10. or 100% sequence identity
  • heavy chain refers to the larger of the two types of polypeptide chains that occur in naturally occurring conformations in antibody molecules and generally determines the class to which the antibody belongs.
  • light chain refers to the smaller of the two types of polypeptide chains that occur in naturally occurring conformations in antibody molecules. Kappa ( ⁇ ) and lambda ( ⁇ ) light chains refer to the two major antibody light chain isotypes.
  • CDR complementarity determining region
  • CDR or FR The precise amino acid sequence of a given CDR or FR may be different due to the different numbering schemes selected, and it should be understood that a "CDR" or "FR" of a given antibody or its region (such as its variable region) covers a CDR or FR defined by any of the above schemes or other known schemes, and in the case where a specified CDR or FR contains a given amino acid sequence, it should be understood that such CDR or FR can also have a sequence of a corresponding CDR or FR defined by any of the above schemes or other known schemes.
  • the numbering scheme used herein to define the boundaries of CDRs and FRs is the Chothia scheme.
  • single-chain antibody refers to a fusion protein comprising at least one light chain variable region (VL) and at least one heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region are adjacent (e.g., connected via a linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • the scFv herein may have the VL and VH in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include VL-linker-VH or VH-linker-VL from the N-terminus to the C-terminus.
  • the term "linker” refers to a molecular sequence connecting two molecules or two sequences on the same molecule.
  • the linker is a peptide linker.
  • the linker does not adversely affect the expression, secretion or biological activity of the polypeptide.
  • the linker is preferably not antigenic and does not induce an immune response.
  • the linker may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a sequence rich in GS) or a non-peptide chemical linker.
  • the linker described in the present invention has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:28 or 29.
  • transmembrane domain refers to a polypeptide structure that enables cell surface molecules to be expressed on the cell surface.
  • the transmembrane domain may be natural or synthetic, and may also be derived from any membrane-bound protein or transmembrane protein. When the antibody binds to the target antigen, the transmembrane domain is capable of signal transduction.
  • Transmembrane domains particularly suitable for use in the present invention can be derived from, for example, TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain is selected from the transmembrane domain of CD8 alpha, CD4, CD28 or CD278.
  • the transmembrane domain is a CD8 alpha or CD28 transmembrane domain, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 12-14.
  • the engineered trophoblast of the present invention can also express a hinge region between the antibody and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antibody.
  • the hinge region can contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region can be derived from all or part of a natural molecule, such as from the extracellular region of all or part of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence.
  • the hinge region is selected from the hinge regions of the following proteins: CD8 ⁇ , CD28, Fc ⁇ RIII ⁇ receptor, IgG4 and IgG1, and more preferably has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:21-24.
  • the cell surface molecule of the present invention further comprises an intracellular domain.
  • the intracellular domain comprises a co-stimulatory domain and/or a primary signaling domain.
  • the primary signaling domain of the present invention can be a cytoplasmic sequence of a T cell receptor and a co-receptor that works together to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar functions.
  • the primary signaling domain comprises two different types of cytoplasmic signal sequences: those that induce antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals.
  • the primary cytoplasmic signal sequence may comprise a number of immunoreceptor tyrosine activation motifs (ITAMs).
  • the primary signaling domain of the present invention includes, but is not limited to, those derived from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, CD66d, and any combination thereof.
  • the primary signaling domain comprises the signaling domain of CD3 ⁇ . More preferably, the primary signaling domain has at least 70%, preferably at least 80%, with the amino acid sequence shown in any one of SEQ ID NOs: 18-20, More preferred is a sequence identity of at least 90%, 95%, 97% or 99% or 100%.
  • the costimulatory domain of the present invention can be an intracellular functional signaling domain from a costimulatory molecule, which can include the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof.
  • “Costimulatory molecule” refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell.
  • Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors.
  • Non-limiting examples of the costimulatory domains of the present invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD270, CD272, CD276, CD278, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and any combination thereof.
  • costimulatory signaling domains derived from the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40,
  • the costimulatory domain is selected from 4-1BB, CD28, CD27, OX40, CD278, or any combination thereof. More preferably, the co-stimulatory domain is 4-1BB or CD28, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:15-17.
  • the cell surface molecule of the present invention further comprises a signal peptide, and the signal peptide is selected from the signal peptides of the following proteins: CD8 ⁇ , IgG1, GM-CSFR ⁇ , IgG4 or any combination thereof.
  • the signal peptide comprises a signal peptide of CD8 ⁇ or B2M. More preferably, the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:25-27.
  • the cell surface molecules of the present invention further comprise a linker for separating any domain/region described herein.
  • the linker may be located between the signal peptide and the antibody, between the VH and VL of the antibody, between the antibody and the hinge region, between the hinge region and the transmembrane domain, on the side of the co-stimulatory domain or on the N- or C- region of the co-stimulatory domain, and/or between the transmembrane domain and the primary signaling domain.
  • the linker may be a peptide of about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length.
  • identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and without considering any conservative substitutions as part of the sequence identity. Alignment to determine the percentage of amino acid sequence identity can be achieved in a variety of ways within the skill of the art using publicly available calculator software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the term “variant” refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence.
  • the variant is a functional variant.
  • the term “functional variant” refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence, and capable of having one or more activities of the reference amino acid sequence.
  • the engineered trophoblast cells of the present invention further express at least one cell activation molecule selected from secreted or membrane-bound cytokines, co-stimulatory ligands, activating receptor agonists and other molecules with cell activation function.
  • the cytokine is selected from secreted or membrane-bound IL-2, IL-5, IL-7, IL-12, IL-15, IL-18, IL-21 and any combination thereof;
  • the co-stimulatory ligand is selected from 4-1BBL, OX40L, B7-H6, CD58, CD112, CD155, MIC-A/B, ULBPs and any combination thereof;
  • the activating receptor agonist is selected from NKG2D agonist, NKp44 agonist, NKp30 agonist, CD16 agonist and any combination thereof;
  • the other molecules with cell activation function include but are not limited to DNER, Brussels, SOM-11, CCN3, MAGP2, MAGP1, TSP2, YB-1, EGFL7, CCR7, DAP12 and DAP10, Notch ligand, etc.
  • the cell activation molecule is selected from IL-21, 4-1BBL, IL-15, IL-21 and 4-1BBL, IL-21 and IL-15, and IL-15 and 4-1BBL, and the IL-21, IL-15 or IL-21 exists in a secretory or membrane-bound form, more preferably IL-21 and 4-1BBL.
  • the present invention provides a cell culture medium comprising the engineered trophoblast cells as described above.
  • the NK cells may be CD56dim NK cells and/or CD56bright NK cells.
  • the NK cells may be derived from primary NK cells, stem cell-derived NK cells, NK cell lines, etc.
  • the primary NK cells may be derived from subjects (such as humans, mice or rats, cats, dogs, cows, horses, sheep, goats or other livestock, etc.), selected from peripheral blood NK cells, umbilical cord blood NK cells, spleen NK cells, etc.
  • the stem cell-derived NK cells may be selected from NK cells derived from pluripotent stem cells (IPSC), NK cells derived from embryonic stem cells (ESC), NK cells derived from hematopoietic stem cells (HSPC), etc.
  • the NK cell line is selected from NK92, NK92.26.5, NK92.MI, NK92Ci, NK92Fc, NK3.3, NKL, NKG, NK-YT, NK-Y
  • the cell is selected from a natural NK cell or an engineered NK cell.
  • the engineered NK cells are selected from NK cells expressing a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor fusion protein (TFP), a T cell antigen coupler (TAC), and an immune mobilizing monoclonal T cell receptor (ImmTAC).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TCP T cell receptor fusion protein
  • TAC T cell antigen coupler
  • ImmTAC immune mobilizing monoclonal T cell receptor
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide, which generally includes an antigen (e.g., tumor antigen) binding domain (e.g., a ligand of an antibody or antigen), a transmembrane domain, an optional co-stimulatory domain, and a primary signaling domain, and each domain is connected by a linker. CAR can redirect the specificity and reactivity of T cells and other immune cells to the selected target in a non-MHC restricted manner.
  • the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain, one or more co-stimulatory domains, and a primary signaling domain.
  • the chimeric antigen receptor also includes one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, etc.
  • a signal peptide a signal peptide
  • a hinge region a suicide gene
  • a switch structure a switch structure
  • T cell receptor refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation.
  • MHC major histocompatibility complex molecule
  • TCR consists of six peptide chains that form heterodimers, which are generally divided into ⁇ type and ⁇ type. Each peptide chain includes a constant region and a variable region, wherein the variable region is responsible for binding to a specific antigen and MHC molecule of specificity.
  • the variable region of TCR may include an antigen binding domain or be operably connected to an antigen binding domain.
  • T cell antigen coupler includes three functional domains: (1) a tumor targeting domain, which may include a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and (3) a transmembrane domain and an intracellular domain of the CD4 co-receptor, wherein the intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR complex as the initial step in T cell activation.
  • TAC immunoreceptor tyrosine-based activation motif
  • T cell receptor fusion protein refers to a recombinant polypeptide derived from various components of TCR, which is usually composed of a TCR subunit and an antigen binding region connected thereto and expressed on the cell surface.
  • the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.
  • Immune mobilizing monoclonal T cell receptor or "ImmTAC” is composed of an engineered T cell receptor (TCR) and an anti-CD3 scFv, wherein: the engineered TCR can specifically bind to the target cell with significantly improved affinity. It specifically recognizes and binds to HLA-peptide complexes on the surface of tumor cells and promotes T cell-mediated effector functions through the interaction of scFv antibody fragments with CD3.
  • the present invention also provides a method for expanding NK cells, comprising: co-culturing NK cells with the engineered trophoblast cells as described above.
  • the time and conditions of co-culture can be selected according to needs and common culture methods in the art.
  • the engineered trophoblast cells are further subjected to irradiation or mitomycin treatment before the co-culturing.
  • Figure 1 Activation and expansion of NK92 cells after treatment with engineered K562 cells.
  • Figure 2 Activation and expansion of sorted PBNK cells after treatment with engineered K562 cells.
  • Figure 3 Activation and expansion of unsorted PBNK cells after treatment with engineered K562 cells.
  • Figure 4 Killing effect of NK92 cells activated by engineered K562 cells on K562 cells.
  • Figure 5 CD107a positivity rate of NK92 cells activated by engineered K562 cells.
  • DNA encoding 4-1BBL (amino acid sequence as shown in SEQ ID NO: 10) was synthesized and cloned into the lentiviral vector pLv-EF1a (i.e., pLVX vector (PPL, Cat No.: PPL00157-4a) in which the promoter is replaced with EF1a) to construct the pLv-EF1a-4-1BBL plasmid.
  • pLv-EF1a i.e., pLVX vector (PPL, Cat No.: PPL00157-4a) in which the promoter is replaced with EF1a
  • DNA encoding mbIL21 (amino acid sequence as shown in SEQ ID NO: 11) was synthesized and cloned into the lentiviral vector pLv-CMV (i.e., pLVX vector (PPL, Cat No.: PPL00157-4a) in which the promoter is replaced with CMV) to construct the pLv-CMV-mbIL21 plasmid.
  • pLv-CMV i.e., pLVX vector (PPL, Cat No.: PPL00157-4a) in which the promoter is replaced with CMV
  • the anti-NKp46 scFv (amino acid sequence as shown in SEQ ID NO: 9), CD8 ⁇ hinge region (amino acid sequence as shown in SEQ ID NO: 22), CD8 ⁇ transmembrane domain (amino acid sequence as shown in SEQ ID NO: 13) were synthesized.
  • the DNA of 147 amino acid sequences was cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-NKp46 CAR plasmid.
  • DNAs encoding anti-NKp46 scFv (amino acid sequence as shown in SEQ ID NO:9), CD8 ⁇ hinge region (amino acid sequence as shown in SEQ ID NO:22), and CD8 ⁇ transmembrane domain (amino acid sequence as shown in SEQ ID NO:13) were synthesized respectively and cloned into the lentiviral vector pLv-EF1a to construct the pLv-EF1a-NKp46 CAR-SHT plasmid.
  • the above plasmids were packaged into lentiviruses, and further transfected into K562 cells to obtain corresponding engineered K562 cells.
  • the plasmids and engineered K562 cells containing them are shown in Table 1. The proportion of positive cells was detected by flow cytometry to determine that each lentivirus was effectively transfected into K562 cells.
  • NKp46 CAR K562 and NKp46 CAR-SHT K562 cells can significantly promote the proliferation of NK92 cells, and there is no significant difference between the two, indicating that NKp46 CAR K562 and NKp46 CAR-SHT K562 both have a significant promoting effect on NK cell expansion, and this effect is not dependent on the intracellular domain.
  • PBMC cells were revived and 10 ml PBMC culture medium (XVIVO-15 + 5% FBS) was added. Centrifuge at 300g for 5 minutes at room temperature and discard the supernatant. Add PBMC culture medium to adjust the cell density to 1 ⁇ 10 6 cells/ml. Place in a cell culture incubator and culture overnight at 37°C. Day 0, the day of activation, transfer the suspended cells in the overnight cultured PBMC to a centrifuge tube, centrifuge at 400g for 5 minutes at room temperature, discard the supernatant, and add magnetic bead sorting buffer. Use the NK cell sorting kit (Miltenyi Biotec, 130-092-657) to separate PBNK.
  • NK cell sorting kit Miltenyi Biotec, 130-092-657
  • Add PBNK culture medium (XVIVO-15+10% FBS+900IU/ml IL2), adjust the PBNK density to 2.5 ⁇ 10 5 cells/ml, and transfer to a cell culture plate. Place in a cell culture incubator and culture at 37°C. Replace half of the medium with PBNK culture medium every 2 to 3 days. Count the cells on the 8th day of culture. The results are shown in Figure 2.
  • PBMC cells were revived and 10 ml PBMC culture medium (XVIVO-15 + 5% FBS) was added. Centrifuge at 300g for 5 minutes at room temperature and discard the supernatant. Add PBMC culture medium to adjust the cell density to 1 ⁇ 10 6 cells/ml. Place in a cell culture incubator and culture overnight at 37°C. On the day of cell activation (Day0), the suspended cells in the overnight cultured PBMC were transferred to a centrifuge tube, centrifuged at 400g for 5 minutes at room temperature, and the supernatant was discarded. Take a portion for flow cytometry analysis and perform absolute counting of PBNK cells. The counting result is used as the starting value of PBNK amplification.
  • PBMC culture medium XVIVO-15 + 5% FBS
  • PBMC cells and Mock K562 (Mock) treated with mitomycin in Example 2.1 4-1BBL + NKp46 CAR K562 (bbl46), NKp46 CAR + mbIL21 K562 (4621), 4-1BBL + NKp46 CAR + mbIL21 K562 (bbl4621) were counted.
  • PBNK culture medium (XVIVO-15 + 10% FBS + 900IU / ml IL2) was added, the PBMC density was adjusted to 6 ⁇ 10 5 / ml, and transferred to a cell culture plate. Place in a cell culture incubator and culture at 37 ° C. Every 2 to 3 days, half of the medium was replaced with PBNK culture medium. Cell counts were performed on the 6th day of culture. The results are shown in Figure 3.
  • Group 1 non-transfected K562 cells (NT), NKp46 CAR K562 (46), NKp46 CAR-SHT K562 (46SHT);
  • Group 2 Mock K562 (Mock), NKp46 CAR K562 (46), NKp46 CAR + mbIL21 K562 (4621), 4-1BBL+NKp46 CAR K562(bbl46), 4-1BBL+NKp46 CAR+mbIL21 K562(bbl4621).
  • NK92 culture medium (XVIVO-15 + 12% FBS + 900 IU/ml IL2) was added, and the cells were placed in a cell culture incubator and cultured at 37°C. Half of the medium was replaced with NK92 culture medium every 2 to 3 days.
  • NK92 cells activated by NKp46 CAR K562 and NKp46 CAR-SHT K562 cells on K562 cells was significantly enhanced, and there was no obvious difference between them, indicating that activating NK cells using NKp46 CAR K562 and NKp46 CAR-SHT K562 cells can enhance the killing effect of NK cells on tumor cells, and this effect is not dependent on the intracellular domain.
  • NK92 cells activated by each engineered K562 cell was significantly enhanced; compared with K562 cells expressing only anti-NKp46 CAR, the additional expression of mbIL21, or 4-1BBL and mbIL21 can further enhance the killing effect of NK92 cells on K562, indicating that the additional expression of some exogenous cell activation molecules on the basis of anti-NKp46 CAR, such as mbIL21, or 4-1BBL and mbIL21, can further enhance the activation effect of NK cells and enhance the tumor cell killing activity of NK cells.
  • Group 1 non-transfected K562 cells (NT), NKp46 CAR K562 (46), NKp46 CAR-SHT K562 (46SHT);
  • Group 2 Mock K562 (Mock), NKp46 CAR K562 (46), NKp46 CAR + mbIL21 K562 (4621), 4-1BBL + mbIL21 K562 (bbl21), 4-1BBL + NKp46 CAR K562 (bbl46), 4-1BBL + NKp46 CAR + mbIL21 K562 (bbl4621).
  • NK92 culture medium (XVIVO-15 + 12% FBS + 900 IU/ml IL2) was added, and the cells were placed in a cell culture incubator and cultured at 37°C. Half of the medium was replaced with NK92 culture medium every 2 to 3 days.
  • NK92 cells One day in advance (Day-1), the culture medium of all NK92 cells was replaced with low-concentration IL-2 culture medium (90 IU/ml IL2).
  • 50 ml of X10 culture medium 45 ml X-VIVO 15 + 5 ml FBS
  • Golgi Stop diluent was prepared: 2 ⁇ l Golgi Stop was added to 3 ml X10 culture medium.
  • NK92 cells and 1 ⁇ 10 5 wild-type K562 cells were added to each well of a 96-well plate, as well as CD107a fluorescent antibodies.
  • the total volume of the cell suspension in each well was 170 ⁇ l, and the cells were incubated at 37°C for 1 hour.
  • 20 ⁇ l of Golgi Stop diluent was added to each well and incubated at 37°C for 2.5 hours.
  • 5 ⁇ l of CD56 fluorescent antibodies were added and incubated at 37°C for 30 minutes.
  • the samples were washed with FACS buffer.
  • the CD56+ cell population of the samples was analyzed by flow cytometry, and the proportion of CD107+ cells was calculated. The results are shown in Figure 5.
  • the CD107a positivity rate of NK cells activated by each engineered K562 cell prepared in the present invention was significantly increased after co-incubation with wild-type K562, and there was little difference between the NK cells activated by each engineered K562 cell, indicating that the cytotoxicity of NK cells activated by each engineered K562 cell expressing a cell surface molecule comprising an antibody targeting NKp46 and a transmembrane domain prepared in the present invention was significantly enhanced.
  • NK cells using engineered trophoblasts expressing antibodies targeting NKp46, cell surface molecules comprising a transmembrane domain and an optional intracellular domain, as well as optional exogenous cell activation molecules (such as 4-1BBL and/or mbIL21, etc.) can significantly enhance the NK cell expansion capacity and tumor cell killing activity.

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Abstract

Trophoblaste modifié, qui exprime une molécule de surface cellulaire reconnaissant spécifiquement NKp46, la molécule de surface cellulaire contenant au moins un anticorps ciblant NKp46 et un domaine transmembranaire. Milieu de culture cellulaire et procédé pour multiplier les cellules NK. Par comparaison avec les trophoblastes non modifiés, le trophoblaste modifié est utilisé pour activer les cellules NK et peut améliorer la capacité de multiplication des cellules NK et l'activité de destruction des cellules tumorales.
PCT/CN2023/100058 2023-06-14 2023-06-14 Trophoblaste modifié et son utilisation Pending WO2024254772A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011110657A1 (fr) * 2010-03-11 2011-09-15 Universite Paris Descartes Détection spécifique de la sous-unité de la gonadotrophine chorionique humaine bêta de type ii produite par les cellules trophoblastiques et néoplasiques
CA3007898A1 (fr) * 2015-12-28 2017-07-06 Innate Pharma Regions variables de proteines se liant a nkp46
CN109666640A (zh) * 2019-01-14 2019-04-23 武汉睿健医药科技有限公司 自然杀伤细胞体外纯培养的方法
CN110300765A (zh) * 2017-01-24 2019-10-01 依奈特制药公司 Nkp46结合剂
CN111206052A (zh) * 2020-02-21 2020-05-29 秦皇岛中邦干细胞医学科技有限公司 一种滋养细胞、其制备方法及其在大量快速扩增γδT细胞中的应用
CN112029001A (zh) * 2020-09-02 2020-12-04 南京北恒生物科技有限公司 靶向nk激活性受体的嵌合抗原受体
CN115595310A (zh) * 2021-07-08 2023-01-13 中国医学科学院血液病医院（中国医学科学院血液学研究所）（Cn） Nk细胞的滋养细胞、其制备方法和应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011110657A1 (fr) * 2010-03-11 2011-09-15 Universite Paris Descartes Détection spécifique de la sous-unité de la gonadotrophine chorionique humaine bêta de type ii produite par les cellules trophoblastiques et néoplasiques
CA3007898A1 (fr) * 2015-12-28 2017-07-06 Innate Pharma Regions variables de proteines se liant a nkp46
CN108779175A (zh) * 2015-12-28 2018-11-09 依奈特制药公司 NKp46结合蛋白的可变区
CN110300765A (zh) * 2017-01-24 2019-10-01 依奈特制药公司 Nkp46结合剂
CN109666640A (zh) * 2019-01-14 2019-04-23 武汉睿健医药科技有限公司 自然杀伤细胞体外纯培养的方法
CN111206052A (zh) * 2020-02-21 2020-05-29 秦皇岛中邦干细胞医学科技有限公司 一种滋养细胞、其制备方法及其在大量快速扩增γδT细胞中的应用
CN112029001A (zh) * 2020-09-02 2020-12-04 南京北恒生物科技有限公司 靶向nk激活性受体的嵌合抗原受体
CN115595310A (zh) * 2021-07-08 2023-01-13 中国医学科学院血液病医院（中国医学科学院血液学研究所）（Cn） Nk细胞的滋养细胞、其制备方法和应用

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